V5-APEX2-SENP2 vector (V000984)

Basic Vector Information

      • Vector Name:
      • V5-APEX2-SENP2
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 10263 bp
      • Type:
      • Mammalian Expression, Lentiviral
      • Promoter:
      • CMV promoter
      • Cloning Method:
      • Gibson Cloning
      • 5' Primer:
      • CMV forward

V5-APEX2-SENP2 vector Vector Map

V5-APEX2-SENP210263 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000pBR322ori-FL4440CAP binding sitelac promoterlac operatorM13 revT3RSV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptidehPGK promoterBSDcPPT/CTSCMV enhancerCMV promoterattB1V5 tagAPEX2WPRE3' LTR (Delta-U3)SV40 poly(A) signalSV40 oriT7M13 Forwardf1 oriAmpR promoterAmpRori

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

V5-APEX2-SENP2 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported               10263 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of APEX2 at nuclear pore.
ACCESSION   .
VERSION     .
KEYWORDS    V5-APEX2-SENP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10263)
  AUTHORS   Fazal FM, Han S, Parker KR, Kaewsapsak P, Xu J, Boettiger AN, Chang 
            HY, Ting AY
  TITLE     Atlas of Subcellular RNA Localization Revealed by APEX-Seq.
  JOURNAL   Cell. 2019 Jun 14. pii: S0092-8674(19)30555-0. doi: 
            10.1016/j.cell.2019.05.027.
  PUBMED    31230715
REFERENCE   2  (bases 1 to 10263)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2019 
            Jun 14. pii: S0092-8674(19)30555-0. doi: 
            10.1016/j.cell.2019.05.027."
FEATURES             Location/Qualifiers
     source          1..10263
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        541..571
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     638..658
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        640..658
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        686..912
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             913..1093
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1140..1265
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1758..1991
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2176..2220
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2395..2895
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2814..2832
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2907..3302
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    3362..3479
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        3596..3899
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3900..4103
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4054..4074
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4126..4150
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4171..4212
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4213..4959
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     misc_feature    6801..7389
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6854..6874)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7272..7283)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7461..7694
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7772..7893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7809..7828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7863..7882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7933..8068
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7995..8014
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(8088..8107)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8089..8107)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8117..8134)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8117..8133)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8126..8148)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8275..8730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8362..8381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8572..8593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8756..8860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             8861..9721
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9079..9098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(9892..10263,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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